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. 2021 May 6;144(4):1152–1166. doi: 10.1093/brain/awab040

Figure 4.

Figure 4

Dietary CLA supplementation in multiple sclerosis patients alters blood myeloid cell composition by enhancing anti-inflammatory and suppressive subsets and functional properties. (A) CD14+ monocytes were isolated from frozen PBMC of RRMS patients and cultured in the presence or absence of 75 µM CLA-mix for 24 h (n = 3). Transcriptional signatures were analysed using the nCounter® Myeloid Innate Immunity Gene Expression Panel (NanoString) and significant differentially expressed genes (P-value < 0.05 and adjusted P-value < 0.5) are illustrated as a heat map. (B) Study design of proof-of-concept trial CLAiMS is shown. (C–G) Frozen PBMC of study subjects at baseline (BL) and 6 months (6M) after CLA supplementation as depicted in B, were stained with monocyte-specific antibodies and analysed by multi-colour flow cytometry. (C) Percentages of CD14+/dim CD16+ (intermediate and non-classical) and CD14+ CD16− (classical) monocytes were evaluated by manual gating (n = 15). (D) Unbiased cluster analysis of monocyte panels is shown as bh-SNE plot of unstimulated monocytes (left) and LPS-stimulated monocytes (right) (n = 12) comparing baseline and 6 months of CLA supplementation. Significantly upregulated clusters are depicted in orange and downregulated clusters are displayed in blue. Clusters with a high (continuous line) and low (dotted line) cytokine expression profile (CCL2, IFNγ, IL1β, IL6, IL8) are highlighted. (E) MFI of pro-inflammatory cytokines in unstimulated CD14+ monocytes, and (F) MFI of CD68 and S100A9 expression in CD14+ CD16− (classical) monocytes were evaluated by manual gating (n = 12). (G) MFI of S100A9 expression in HLA-DRlow/−CD14+ cells (MDSC-like phenotype) (n = 12) is shown. (H) Transcriptional signatures of CD14+ monocytes isolated at baseline and after 6 months of CLA supplementation were analysed by the nCounter® Myeloid Innate Immunity Gene Expression Panel (NanoString) and illustrated as a heat map (n = 12, P-value < 0.05). (I) Oxygen consumption rate of CD14+ monocytes isolated at BL and after 6M of CLA supplementation is shown for basal and maximal respiration (n ≥ 10). Dot plots depict mean ± SD. Each dot represents one individual patient at baseline or 6 months; in E–G only, dots belonging to the same patient are connected by a line. Statistics: paired two-tailed Student’s t-test; reg. = regulated.